A certain kind of peptides such as γ-glutamylvalylglycine (L-γ-glutamyl-L-valyl-glycine, henceforth referred to as “γ-Glu-Val-Gly”) have a calcium receptor agonistic activity (Patent document 1). Such peptides having a calcium-receptor activation action are known to be able to impart kokumi taste to foods and drinks (Patent document 2), improve tastes of low fat diets, especially fat-like thickness and smoothness (Patent document 3), improve feeling of body of sweet taste substances, and improve bitterness peculiar to sweet taste substances (Patent document 4).
Moreover, such peptides as described above are known to have a prophylactic or curative effect on diarrhea (Patent document 5) and diabetes (Patent document 6), and a bicarbonate secretion promoting effect in the alimentary tract (Patent document 7).
As described above, wide application of γ-Glu-Val-Gly in the field of food, drug, and so forth is expected.
As methods for producing tripeptides, chemical synthesis methods and enzymatic methods are generally known. As one of the chemical synthesis methods, a method of selectively γ-glutamylating a dipeptide by using N-protected glutamic anhydride to obtain a tripeptide is known (Patent document 10). As the enzymatic methods, there is known, for example, a method of reacting a dipeptide having an esterified or amidated carboxyl terminus and an amino acid having free amino group (for example, an amino acid of which carboxyl group is protected) in the presence of a peptide-producing enzyme to produce a tripeptide (Patent document 8).
As an enzyme that catalyzes the reaction of transferring γ-glutamyl group to a dipeptide, γ-glutamyl transferase (also called γ-glutamyl transpeptidase, henceforth also referred to as “GGT”) is known. It was reported that in a reaction of Val-Gly (valylglycine) and γ-glutamyl-p-nitroanilide as a glutamine donor in the presence of that enzyme, the activity of the enzyme was detected by color development of p-nitroaniline (Non-patent document 1), but generation of γ-Glu-Val-Gly was not confirmed.
GGT of Escherichia coli consists of two subunits, a large subunit and a small subunit, and the ggt gene coding for GGT comprises ORFs (open reading frames) coding for a leader peptide, the large subunit, and the small subunit (Patent document 9). With advance of the transcription and translation of the ggt gene, transfer of the translation product to the periplasm, cleavage of the leader peptide and processing for generating the large subunit and the small subunit occur to generate the mature GGT.
As for researches concerning the structural analysis of GGT, it was reported that the D433N mutation (Non-patent document 2) or the Y444 mutation and G484 mutation (Non-patent document 3) of Escherichia coli GGT impart a novel acylase activity, specifically an ability to hydrolyze glutaryl-7-aminocephalosporanic acid, to GGT, or improve such an ability. Non-patent document 3 (Yamada et al.) specifically describes N411G, N411H, N411Y, Q430I, Q430V, D433A, D433G, D433L, D433N, Y444A, Y444F, Y444G, Y444H, Y444I, Y444L, Y444V, G484A, P460V, L461F, and S462T mutations.
Further, as information concerning the structure around the active center of GGT, it was reported that Y444 in GGT of Escherichia coli (Non-patent document 3) or Y433 in GGT of Helicobacter Pylori (Non-patent document 4) locates in the lid-loop that covers the substrate-binding site.
Furthermore, it was also reported that Arg114 and Arg337 are important for the function of GGT of Escherichia coli (Non-patent document 5), and this reference describes R114K, R114L, R114D, R337K, R337L, and R337D mutations.
Further, it is also reported that most of mutants of Escherichia coli GGT having mutations at Thr407, Asp433, and Met464 in the small subunit lost the activity (Non-patent document 6). This reference describes N411G, N411H, N411Y, Q430I, Q430V, D433A, D433G, D433L, D433N, Y444A, Y444F, Y444G, Y444H, Y444I, Y444L, Y444V, G484A, P460V, L461F, and S462T mutations.
In addition, influences of T391, T392, H393, Q390 and V396 mutations (Non-patent document 7), and R513 and R571 mutations (Non-patent document 8) on processing into the subunits of Escherichia coli GGT or the GGT activity were reported. The former reference describes T391A, T391S, T392A, H393G, Q390A, and V396T mutations, and the latter reference describes R513A, R513G, R571A, and R571G mutations.
However, any mutation or combination of mutations preferred for the γ-glutamylation of Val-Gly is not known.
For the decomposition of Val-Gly, it was reported that PepA hydrolyzes Val-Gly (Non-patent document 9), and PepA- and PepN-deficient Escherichia coli does not decompose Val-Gly (Non-patent document 10), but activity of pepD for decomposing Val-Gly is not known.